Fanny Legrand1, Yun Cao2, Joshua B Wechsler3, Xiang Zhu4, Nives Zimmermann4, Shakuntala Rampertaap5, Joseph Monsale5, Kimberly Romito5, Bradford A Youngblood6, Emily C Brock6, Michelle A Makiya7, Nenad Tomasevic6, Christopher Bebbington6, Irina Maric8, Dean D Metcalfe9, Bruce S Bochner2, Amy D Klion10. 1. Human Eosinophil Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Electronic address: fanny.legrand@nih.gov. 2. Division of Allergy and Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Ill. 3. Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, Ill. 4. Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio. 5. Department of Laboratory Medicine, Warren Magnusson Clinical Center, National Institutes of Health, Bethesda. 6. Allakos, Inc, San Carlos, Calif. 7. Human Eosinophil Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md. 8. Hematology Section, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda. 9. Mast Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md. 10. Human Eosinophil Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Electronic address: aklion@nih.gov.
Abstract
BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.
BACKGROUND:Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS:Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS:Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.
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